3-aminopyrazole inhibitors of cyclin dependent kinases

ABSTRACT

The present invention describes compounds of formula I                    
     and pharmaceutically acceptable salts thereof. 
     The formula I compounds are protein kinase inhibitors and are useful in the treatment of proliferative diseases, for example, cancer, inflammation and arthritis. They may also be useful in the treatment of Alzheimer&#39;s disease, and cardiovascular disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application claims priority from provisional U.S.application Ser. No. 60/180,609, filed Feb. 7, 2000, incorporated hereinby reference in its entirety.

BRIEF DESCRIPTION OF THE INVENTION

The present invention is directed to compounds of the formula

and pharmaceutically acceptable salts thereof. As used in formula I, andthroughout the specification, the symbols have the following meanings:

R¹=R², COR³, CONH₂, CONR²R³, COOR³, or SO₂R³;

R²=H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, orheteroarylium;

R³=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl;

 where i, j=0 or 1 but cannot both be 1, and Y=optionally substitutedethylene, alkene, alkyne, or any 2 adjacent carbon atoms of a cycloalkylor cycloheteroalkyl ring of 3-7 atoms;

R⁴=alkyl of 2 or more carbon atoms, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, heterocycloalkylalkyl, alkyloxycarbonyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, aryloxycarbonyl, or R⁹, providedthat R⁴ is other than an optionally substituted phenyl, pyridyl,pyrimidyl, pyrazinyl, pyridazinyl or piperidinyl ring;

R⁵, R⁶, R⁷, R⁸=independently H, alkyl, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, halo, or hydroxy, alkoxy, amino, NR¹⁵R¹⁶, thio, oralkylthio with the proviso that only one such heteroatom group is bondedto any one carbon atom;

 where Z=O, NR¹², or S;

R¹⁰, R¹¹=independently H, alkyl, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, halo, hydroxy, alkoxy, alkylcarbonyloxy, carboxy,alkyloxycarbonyl, amino, NR¹³R¹⁴, carbamoyl, ureido, thio, or alkylthio;and

R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶=independently H, alkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, or heteroarylalkyl.

The compounds of formula I are protein kinase inhibitors and are usefulin the treatment of proliferative diseases, for example, cancer,inflammation and arthritis. They may also be useful in the treatment ofAlzheimer's disease, and cardiovascular disease.

DESCRIPTION OF THE INVENTION

The present invention provides for compounds of formula I,pharmaceutical compositions employing such compounds, and for methods ofusing such compounds.

Listed below are definitions of various terms used to describe thecompounds of the instant invention. These definitions apply to the termsas they are used throughout the specification (unless they are otherwiselimited in specific instances) either individually or as part of alarger group.

It should be noted that any heteroatom with unsatisfied valences isassumed to have hydrogen atoms sufficient to satisfy the valences.

Carboxylate anion refers to a negatively charged group —COO⁻.

The term “alkyl” or “alk” refers to a monovalent alkane (hydrocarbon)derived radical containing from 1 to 12 carbon atoms unless otherwisedefined. An alkyl group is an optionally substituted straight, branchedor cyclic saturated hydrocarbon group. When substituted, alkyl groupsmay be substituted with up to four substituent groups, R as defined, atany available point of attachment. When the alkyl group is said to besubstituted with an alkyl group, this is used interchangeably with“branched alkyl group”. Exemplary unsubstituted such groups includemethyl, ethyl, propyl, isopropyl, n-butyl, t-butyl, isobutyl, pentyl,hexyl, isohexyl, heptyl, 4,4-dimethylpentyl, octyl,2,2,4-trimethylpentyl, nonyl, decyl, undecyl, dodecyl, and the like.Exemplary substituents may include but are not limited to one or more ofthe following groups: halo (such as F, Cl, Br, I), haloalkyl (such asCCl₃ or CF₃), alkoxy, alkylthio, hydroxy, carboxy (—COOH),alkyloxycarbonyl (—COOR), alkylcarbonyloxy (—OCOR), amino (—NH₂),carbamoyl (—NHCOOR— or —OCONHR—), urea (—NHCONHR—), amidinyl (—CNHNHR or—CNRNH₂), or thiol (—SH). Alkyl groups as defined may also comprise oneor more carbon to carbon double bonds or one or more carbon to carbontriple bonds.

The term “alkenyl” refers to a hydrocarbon radical straight, branched orcyclic containing from 2 to 12 carbon atoms and at least one carbon tocarbon double bond.

The term “alkynyl” refers to a hydrocarbon radical straight, branched orcyclic, containing from 2 to 12 carbon atoms and at least one carbon tocarbon triple bond.

The term “ethylene” refers to a —CH₂CH₂— group.

Cycloalkyl is a specie of alkyl containing from 3 to 15 carbon atoms,without alternating or resonating double bonds between carbon atoms. Itmay contain from 1 to 4 rings. Exemplary unsubstituted such groupsinclude cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, etc. Exemplarysubstituents include one or more of the following groups: halogen,alkyl, alkoxy, alkyl hydroxy, amino, nitro, cyano, thiol and/oralkylthio.

The terms “alkoxy” or “alkylthio”, as used herein, denote an alkyl groupas described above bonded through an oxygen linkage (—O—) or a sulfurlinkage (—S—), respectively.

Sulfoxide and sulfone denote groups bonded by —SO— or —SO₂— linkages,respectively.

The term “alkyloxycarbonyl”, as used herein, denotes an alkoxy groupbonded through a carbonyl group. An alkoxycarbonyl radical isrepresented by the formula: —C(O)OR, where the R group is a straight orbranched C₁₋₆ alkyl group.

The term “alkylcarbonyl” refers to an alkyl group bonded through acarbonyl group.

The term “alkylcarbonyloxy”, as used herein, denotes an alkylcarbonylgroup which is bonded through an oxygen linkage.

The term “arylalkyl”, as used herein, denotes an aromatic ring bonded toan alkyl group as described above.

The term “aryl” refers to monocyclic or bicyclic aromatic rings, e.g.phenyl, substituted phenyl and the like, as well as groups which arefused, e.g., naphthyl, phenanthrenyl and the like. An aryl group thuscontains at least one ring having at least 6 atoms, with up to five suchrings being present, containing up to 22 atoms therein, with alternating(resonating) double bonds between adjacent carbon atoms or suitableheteroatoms. Aryl groups may optionally be substituted with one or moregroups including, but not limited to halogen, alkyl, alkoxy, hydroxy,carboxy, carbamoyl, alkyloxycarbonyl, nitro, trifluoromethyl, amino,cycloalkyl, cyano, alkyl S(O)_(m)(m=O, 1, 2), or thiol.

The term “heteroaryl” refers to a monocyclic aromatic hydrocarbon grouphaving 5 or 6 ring atoms, a bicyclic aromatic group having 8 to 10atoms, or a tricyclic aromatic group having 10 to 16 atoms, containingat least one heteroatom, O, S, or N, in which a carbon or nitrogen atomis the point of attachment, and in which one or two carbon atom(s) isoptionally replaced by a heteroatom selected from O or S, or in whichfrom 1 to 3 additional carbon atoms are optionally replaced by nitrogenheteroatoms, said heteroaryl group being optionally substituted asdescribed herein. Exemplary heteroaryl groups include the following:thienyl, furyl, pyrrolyl, pyridinyl, imidazolyl, pyrrolidinyl,piperidinyl, thiazolyl, oxazolyl, triazolyl, pyrazolyl, isoxazolyl,isothiazolyl, pyrazinyl, tetrazolyl, pyridazinyl, pyrimidinal,triazinylazepinyl, indolyl, isoindolyl, quinolinyl, isoquinolinyl,benzothiazolyl, benzoxazolyl, benzimidazolyl, benzoxadiazolyl,benzofurazanyl and tetrahydropyranyl. Exemplary substituents mayinclude, but are not limited to, one or more of the following: halogen,alkyl, alkoxy, hydroxy, carboxy, carbamoyl, alkyloxycarbonyl,trifluoromethyl, cycloalkyl, nitro, cyano, amino, alkylS(O)_(m)(m=0, 1,2), or thiol.

The term “heteroarylium” refers to heteroaryl groups bearing aquaternary nitrogen atom and thus a positive charge.

The term “heterocycloalkyl” refers to a cycloalkyl group (nonaromatic)in which one of the carbon atoms in the ring is replaced by a heteroatomselected from O, S or N, and in which up to three additional carbonatoms may be replaced by said heteroatoms.

The term “quaternary nitrogen” refers to a tetravalent positivelycharged nitrogen atom including, e.g. the positively charged nitrogen ina tetraalkylammonium group (e.g. tetramethylammonium,N-methylpyridinium), the positively charged nitrogen in protonatedammonium species (e.g. trimethylhydroammonium, N-hydropyridinium), thepositively charged nitrogen in amine N-oxides (e.g.N-methyl-morpholine-N-oxide, pyridine-N-oxide), and the positivelycharged nitrogen in an N-amino-ammonium group (e.g. N-aminopyridinium).

The term “heteroatom” means O, S or N, selected on an independent basis.

The term “halogen” or “halo” refers to chlorine, bromine, fluorine oriodine.

When a functional group is termed “protected”, this means that the groupis in modified form to preclude undesired side reactions at theprotected site. Suitable protecting groups for the compounds of thepresent invention will be recognized from the present application takinginto account the level of skill in the art, and with reference tostandard textbooks, such as Greene, T. W. et al., Protective Groups inOrganic Synthesis, Wiley, N.Y. (1991).

Suitable examples of salts of the compounds according to the inventionwith inorganic or organic acids are hydrochloride, hydrobromide,sulfate, phosphate. Salts which are unsuitable for pharmaceutical usesbut which can be employed, for example, for the isolation orpurification of free compounds I or their pharmaceutically acceptablesalts, are also included.

All stereoisomers of the compounds of the instant invention arecontemplated, either in admixture or in pure or substantially pure form.The definition of the compounds according to the invention embraces allpossible stereoisomers and their mixtures. It very particularly embracesthe racemic forms and the isolated optical isomers having the specifiedactivity. The racemic forms can be resolved by physical methods, suchas, for example, fractional crystallization, separation orcrystallization of diastereomeric derivatives or separation by chiralcolumn chromatography. The individual optical isomers can be obtainedfrom the racemates by conventional methods, such as, for example, saltformation with an optically active acid followed by crystallization.

All configurational isomers of compounds of the present invention arecontemplated, either in admixture or in pure or substantially pure form.The definition of compounds of the present invention very particularlyembraces both cis (Z) and trans (E) alkene isomers, as well as cis andtrans isomers of cycloalkyl or heterocycloalkyl rings.

Furthermore, with regard to the object compound I, it is to beunderstood that the compound includes tautomeric isomers. That is thecompound of formula I may exist in either of the the following forms:

in a state of equilibrium.

It should be understood that solvates (e.g. hydrates) of the compoundsof formula I are also within the scope of the present invention. Methodsof solvation are generally known in the art. Accordingly, the compoundsof the instant invention may be in the free or hydrate form, and may beobtained by methods exemplified by the following schemes.

As illustrated in Scheme I, a compound of formula III may be prepared bytreating a solution of 5-nitro-3-pyrazole carboxylic acid (II) inabsolute ethanol with H₂SO₄ to form the ester. The compound of formulaIV may be prepared by treating nitro compound III with a reducing agentsuch as hydrogen in the presence of a catalyst such as palladium oncharcoal, or with a metal reducing agent such as tin or iron. The aminogroup and one of the pyrazole ring nitrogen atoms are protected bytreating a compound of formula IV with di-tert butyl dicarbonate or acomparable carbamylating agent and a base such as pyridine. The compondof formula V may be reduced with lithium aluminum hydride or acomparable reducing agent to give the alcohol VI, which may be oxidizedby manganese dioxide or a comparable oxidizing agent to give an aldehydeof formula VII. The aldehyde of formula VII may be reacted with aphosphonate ester and a base such as potassium t-butoxide to provide anolefin of formula VIII. The olefin of formula VIII may be either cis(Z), trans (E) or a mixture of the two isomers. In the event that thecompound of formula VIII is a single isomer, it may be equilibrated withits geometric olefin isomer by treatment with an agent such asultraviolet light (UV) and acetone. In the specific instance illustratedthe E alkene isomer VIII is interconverted with the Z alkene isomer IX.A compound of formula IX may be deprotected by treating with an acidsuch as trifluoroacetic acid or hydrochloric acid in a solvent such asdioxane to give the free amine X, which may be reacted with an acylatingagent such as an isocyanate, acid anhydride, acid chloride, or the like,to give a compound of formula XI, which is a compound of formula I whereR¹ is CONR²R³ and Y is Z alkenyl.

According to Scheme II, a solution of 5-ethylcarboxylate-3-aminopyrazoleIV, and a base such as pyridine may be reacted with an acid chloridesuch as 2-methyl butanoyl chloride to give the compound of formula XII.Treatment of a compound of formula XII with a reducing agent such aslithium aluminum hydride (LAH) in tetrahydrofuran (THF) gives thealcohol of formula XIII, which may be oxidized by an oxidant such asmanganese dioxide in acetone to give an aldehyde of formula XIV.Aldehydes of formula XIV may be treated with a phosphonate ester in thepresence of a base such as potassium tert-butoxide to give compounds offormula XV. Analogous to Scheme I, compounds of formula XV(which is acompound of formula I where Y is an E alkene) may be treated withultraviolent light and acetone to give a compound of formula XVI (whichis a compound of formula I where R¹ is COR³ and Y is Z alkenyl).

Additional compounds of formula I where Y is an alkyne may be preparedby methods known to those of ordinary skill in the art. A compound offormula I where Y is an alkene may be reacted with a reducing agent suchas hydrogen in the presence of a catalyst such as palladium on carbon toprovide a compound of formula I where Y is ethylene. Compounds offormula I where Y is an alkyne may be prepared from the compound offormula I where Y is an alkene by methods known in the art, such ashalogenation and dehydrohalogenation.

The starting compounds of Schemes I and II are commercially available ormay be prepared by methods known to one of ordinary skill in the art.

The compounds shown in Schema I and II are illustrative, and the methodsdescribed may be used by those of ordinary skill in the art to preparethe analogous intermediates encompassed by the genus. All compounds offormula I may be prepared by modification of the procedures describedherein.

A first group of preferred compounds of formula I are those where:

R¹=R², COR³, or CONR²R³;

R²=H, alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl;

R³=alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl;

 where i=j=0;

Y=optionally substituted ethylene, alkene, or any two adjacent carbonatoms of a cycloalkyl ring;

R⁴=alkyl of two or more carbon atoms, aryl, heteroaryl, or R⁹;

R⁵, R⁶, R⁷, R⁸=independently H, or alkyl;

 where Z=O; and

R¹⁰, R¹¹=independently H, or alkyl.

A second group of preferred compounds of formula I are those where:

R¹=CONR²R³;

R²=H, alkyl, heteroaryl, arylalkyl, or heteroarylalkyl;

R³=aryl;

 where i, j=0 or 1 but cannot both be 1, and Y=an optionally substitutedethylene or alkene;

R⁴=R⁹;

R⁵, R⁶, R⁷, R⁸=independently H, or alkyl;

 where Z=O; and

R¹⁰, R¹¹=independently H, or alkyl.

A third group of preferred compounds of formula I are those where:

R¹=R²;

R²=alkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl;

 where i, j=0 or 1 but cannot both be 1, and Y=an optionally substitutedethylene or alkene;

R⁵, R⁶, R⁷, R⁸=independently H, or alkyl;

R⁴=R⁹;

 where Z=O;

R¹⁰=alkyl; and

R¹¹=H.

A fourth group of preferred compounds of formula I are those where:

R¹=COR³;

R³=alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl;

 where i, j=0 or 1 but cannot both be 1, and Y=an optionally substitutedethylene, alkene or alkyne;

R⁴=R⁹;

R⁵, R⁶, R⁷, R⁸=independently H, or alkyl;

 where Z=O;

R¹⁰=alkyl; and

R¹¹=H.

Formula I compounds particularly useful in the methods of this inventioninclude:

N,N-[(Z)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylamine;

N-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide;

N-(Z)-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide;and

N,N-[(E)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylamine, among others.

The compounds according to the invention have pharmacologicalproperties; in particular, the compounds of formula I are inhibitors ofprotein kinases such as the cyclin dependent kinases (cdks), forexample, cdc2 (cdk1), cdk2, and cdk4. The novel compounds of formula Iare expected to be useful in the therapy of proliferative diseases suchas cancer, inflammation, arthritis, Alzheimer's disease andcardiovascular disease. These compounds may also be useful in thetreatment of topical and systemic fungal infections.

More specifically, the compounds of formula I are useful in thetreatment of a variety of cancers, including (but not limited to) thefollowing:

carcinoma, including that of the bladder, breast, colon, kidney, liver,lung, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin;

hematopoietic tumors of lymphoid lineage, including acute lymphocyticleukemia, B-cell lymphoma, and Burkett's lymphoma;

hematopoietic tumors of myeloid lineage, including acute and chronicmyelogenous leukemias and promyelocytic leukemia;

tumors of mesenchymal origin, including fibrosarcoma andrhabdomyosarcoma; and

other tumors, including melanoma, seminoma, teratocarcinoma,osteosarcoma, neuroblastoma and glioma.

Due to the key role of cdks in the regulation of cellular proliferationin general, inhibitors could act as reversible cytostatic agents whichmay be useful in the treatment of any disease process which featuresabnormal cellular proliferation, e.g., neuro-fibromatosis,atherosclerosis, pulmonary fibrosis, arthritis, psoriasis,glomerulonephritis, restenosis following angioplasty or vascularsurgery, hypertrophic scar formation, inflammatory bowel disease,transplantation rejection, angiogenesis, and endotoxic shock.

Compounds of formula I may also be useful in the treatment ofAlzheimer's disease, as suggested by the recent finding that cdk5 isinvolved in the phosphorylation of tau protein (J. Biochem, 117, 741-749(1995)).

Compounds of formula I may also act as inhibitors of other proteinkinases, e.g., protein kinase C, her2, rafI, MEK1, MAP kinase, EGFreceptor, PDGF receptor, IGF receptor, PI3 kinase, wee I kinase, Src,Abl, VEGF, and 1ck, and thus be effective in the treatment of diseasesassociated with other protein kinases.

Compounds of formula I also induce or inhibit apoptosis, a physiologicalcell death process critical for normal development and homeostasis.Alterations of apoptotic pathways contribute to the pathogenesis of avariety of human diseases. Compounds of formula I, as modulators ofapoptosis, will be useful in the treatment of a variety of humandiseases with abberations in apoptosis including cancer (particularly,but not limited to follicular lymphomas, carcinomas with p53 mutations,hormone dependent tumors of the breast, prostate and ovary, andprecancerous lesions such as familial adenomatous polyposis), viralinfections (including but not limited to herpesvirus, poxvirus,Epstein-Barr virus, Sindbis virus and adenovirus), autoimmune diseases(including but not limited to systemic lupus, erythematosus, immunemediated glomerulonephritis, rheumatoid arthritis, psoriasis,inflammatory bowel diseases, and autoimmune diabetes mellitus),neurodegenerative disorders (including but not limited to Alzheimer'sdisease, AIDS-related dementia, Parkinson's disease, amyotrophic lateralsclerosis, retinitis pigmentosa, spinal muscular atrophy and cerebellardegeneration), AIDS, myelodysplastic syndromes, aplastic anemia,ischemic injury associated myocardial infarctions, stroke andreperfusion injury, arrhythmia, atherosclerosis, toxin-induced oralcohol induced liver diseases, hematological diseases (including butnot limited to chronic anemia and aplastic anemia), degenerativediseases of the musculoskeletal system (including but not limited toosteoporosis and arthritis), aspirin-sensitive rhinosinusitis, cysticfibrosis, multiple sclerosis, kidney diseases, and cancer pain.

The compounds of this invention may also be useful in combination withknown anti-cancer treatments such as radiation therapy or withcytostatic and cytotoxic agents, such as for example, but not limitedto, DNA interactive agents, such as cisplatin or doxorubicin; inhibitorsof farnesyl protein transferase, such as those described in pending U.S.application Ser. No. 08/802,329, topoisomerase II inhibitors, such asetoposide; topoisomerase I inhibitors, such as CPT-11 or topotecan;tubulin stabilizing agents, such as paclitaxel, docetaxel or theepothilones; hormonal agents, such as tamoxifen; thymidilate synthaseinhibitors, such as 5-fluorouracil; and antimetabolites, such asmethoxtrexate; antiangiogenic agents, such as angiostatin or endostatin;and kinase inhibitors, such as her2 specific antibodies. The formula Icompounds of this invention may also be useful in combination withmodulators of p53 transactivation. In addition, the formula I compoundsmay be used for treating chemotherapy-induced alopecia,chemotherapy-induced thrombocytopenia, chemotherapy-induced leukopeniaor mucocitis. In the treatment of chemotherapy-induced alopecia, theformula I compound is preferably topically applied in the form of amedicament such as a gel, solution, dispersion or paste.

If formulated as a fixed dose, such combination products employ thecompounds of this invention within the dosage range described below andthe other pharmaceutically active agent within its approved dosagerange. For example, the cdc2 inhibitor olomucine has been found to actsynergistically with known cytotoxic agents in inducing apoptosis (J.Cell Sci., 108, 2897 (1995)). Compounds of formula I may be usedsequentially with known anti-cancer or cytotoxic agents when acombination formulation is inappropriate.

cdc2/cyclin B1 Kinase Assay

cdc2/cyclin B1 kinase activity was determined by monitoring theincorporation of ³²P into histone HI. The reaction consisted of 50 ngbaculovirus expressed GST-cdc2, 75 ng baculovirus expressed GST-cyclinB1, 1 μg histone HI (Boehringer Mannheim), 0.2 μCi of ³²P γ-ATP and 25μM ATP in kinase buffer (50 mM Tris, pH 8.0, 10 mM MgCl₂, 1 mM EGTA, 0.5mM DTT). The reaction was incubated at 30° C. for 30 minutes and thenstopped by the addition of cold trichloroacetic acid (TCA) to a finalconcentration of 15% and incubated on ice for 20 minutes. The reactionwas harvested onto GF/C unifilter plates (Packard) using a PackardFiltermate Universal harvester, and the filters were counted on aPackard TopCount 96-well liquid scintillation counter (Marshak, D. R.,Vanderberg, M. T., Bae, Y. S., Yu, I. J., J. of Cellular Biochemistry,45, 391-400 (1991), incorporated by reference herein).

cdk2/cyclin E Kinase Assay

cdk2/cyclin E kinase activity was determined by monitoring theincorporation of ³²P into the retinoblastoma protein. The reactionconsisted of 2.5 ng baculovirus expressed GST-cdk2/cyclin E, 500 ngbacterially produced GST-retinoblastoma protein (aa 776-928), 0.2 μCi³²P γ-ATP and 25 μM ATP in kinase buffer (50 mM Hepes, pH 8.0, 10 mMMgCl₂, 5 mM EGTA, 2 mM DTT). The reaction was incubated at 30° C. for 30minutes and then stopped by the addition of cold trichloroacetic acid(TCA) to a final concentration of 15% and incubated on ice for 20minutes. The reaction was harvested onto GF/C unifilter plates (Packard)using a Packard Filtermate Universal harvester, and the filters werecounted on a Packard TopCount 96-well liquid scintillation counter.

cdk4/cyclin D1 Kinase Activity

cdk4/cyclin D1 kinase activity was determined by monitoring theincorporation of ³²P in to the retinoblastoma protein. The reactionconsisted of 165 ng baculovirus expressed as GST-cdk4, 282 ngbacterially expressed as S-tag cyclin D1, 500 ng bacterially producedGST-retinoblastoma protein (aa 776-928), 0.2 μCi ³²P γ-ATP and 25 μM ATPin kinase buffer (50 mM Hepes, pH 8.0, 10 mM MgCl₂, 5 mM EGTA, 2 mMDTT). The reaction was incubated at 30° C. for 1 hour and then stoppedby the addition of cold trichloroacetic acid (TCA) to a finalconcentration of 15% and incubated on ice for 20 minutes. The reactionwas harvested onto GF/C unifilter plates (Packard) using a PackardFiltermate Universal harvester, and the filters were counted on aPackard TopCount 96-well liquid scintillation counter (Coleman, K. G.,Wautlet, B. S., Morissey, D, Mulheron, J. G., Sedman, S., Brinkley, P.,Price, S., Wedster, K. R. (1997) Identification of CDK4 Sequencesinvolved in cyclin D, and p16 binding. J. Biol. Chem.272,30:18869-18874, incorporated by reference herein).

The following examples and preparations describe the manner and processof making and using the invention and are illustrative rather thanlimiting. It should be understood that there may be other embodimentswhich fall within the spirit and scope of the invention as defined bythe claims appended hereto.

EXAMPLE 1N,N-[(Z)-5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylAmine (XI)

A. Preparation of 5-Nitro-3-pyrazole-ethylcarboxylate (III)

A solution of 10 g of 5-nitro-3-pyrazole carboxylic acid was heated toreflux in 50 ml of absolute ethanol and 2 ml of H₂SO₄. A Dean Stark trapwas used to azeotrope the water produced. The solvent was removed undervacuum to give a white solid, which was used in the next step withoutfurther purification.

B. Preparation of 5-Carboxyethyl-3-aminopyrazole (IV)

To a Parr bottle was added 300 mg of Pd/C (10%), 3 g of III and 125 mlof absolute ethanol. The hydrogenation was run at 30 psi for 4 hours,the catalyst removed by filtration through a cake of celite and the cakewas washed with 30 ml of ethanol. To the ethanol solution was added 1 mlof HCl and then the solvent was taken to dryness under vacuum and theproduct was used in the next step without further purification.

C. Preparation of5-Carboxyethyl-1,3-bis[1,1-dimethyl(ethyl)oxycarbonyl]-3-aminopyrazole(V)

To a solution of IV in 50 ml of anhydrous pyridine was added 10.9 g ofdi-tertbutyldicarbonate. The reaction was heated to 95° C. for 12 hoursand then the excess pyridine was removed under vacuum. The gum was takenup into 50 ml of pyridine and 11 g of di-tertbutyldicarbonate was added.The reaction was stirred for 1.5 hr at room temperature. The solvent wasremoved under vacuum and the orange/brown gum was dried under highvacuum overnight, and used in the next step without furtherpurification.

D. Preparation of5-Hydroxymethyl-3-[1,1-dimethyl(ethyl)oxycarbonyl]aminopyrazole (VI)

A solution consisting of 4.46 g of V in 100 ml of dry THF under a streamof N₂ was cooled to 0° C. with an ice bath. While keeping thetemperature at 0° C., 37.69 ml of 1 M LiAlH₄ in THF was added to the THFsolution over a period of 60 minutes. After stirring for 2 hours at 0°C., the solution was poured over 300 g of crushed ice. Once the ice hadmelted, the solution was brought to pH 7 by addition of HCl. The waterlayer was filtered to remove aluminum salts and the filtrate wasextracted three times with 200 ml of ethyl acetate. The organic layerwas collected, dried over MgSO₄ and evaporated to an orange solid. Thesolid was purified by silica gel chromatography to provide a total of1.7 g of the alcohol (63%).

E. Preparation of5-Carboxaldehyde-3-[1,1-dimethyl(ethyl)oxycarbonyl]aminopyrazole (VII)

A solution consisting of 1.7 g of VI, 2.08 g of MnO₂ in 30 ml of acetonewas heated at reflux for 24 hours. An additional 500 mg of MnO₂ wasadded and the reaction heated to reflux for an additional 3 hours. Themixture was filtered through a cake of Celite and the cake was washedwith excess acetone. The solvent was removed to yield a brown solid,which was purified by crystallization from dichloromethane to provide960 mg (57%) of the desired product in pure form.

F. Preparation ofN,N-[5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]1,1-dimethyl(ethyl)oxycarbonylAmine (VIII)

A solution of (5-t-butyl-oxazol-2-yl)methylphosphonic acid diethyl ester(0.88 g) in 10 ml of anhydrous THF under N₂ was cooled to 0° C. and 1.4g of potassium tertbutoxide was added. The reaction was allowed to stirat 0° C. for 30 minutes at which time the anion solution was syringedinto a solution of 960 mg of VII dissolved in 35 ml of anhydrous THFunder N₂. The reaction was allowed to stir at room temperature for 1hour at which point an additional portion of the anion solution (230 mgof the phosphonic acid ethyl ester mixed with 368 mg of potassiumtertbutoxide as above) was added and the reaction was allowed to stirovernight at room temperature. The solvent was removed under vacuum togive an orange solid and the desired product was purified by silica gelchromatography to give 1.2 g (93%) of the desired product as a tansolid. (M+H)⁺=333. HPLC RT=4.17 min (YMC S5 ODS column, 4.6×50 mm;10-90% MeOH/H₂O gradient,+0.1% TFA; 4 ML/min, 220 nM detection)

G. Preparation ofN,N-[(Z)-5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]1,1-dimethyl(ethyl)oxycarbonylAmine (IX)

Into a jacketed 400 watt Hanovia mercury lamp was placed 312 mg of VIIIin 20 ml of acetone and the reaction was irradiated for 24 hours toyield 82% of the desired Z isomer. The solvent was removed and the crudematerial was used in the next step.

H. Preparation of3-Amino-5-[(Z)-5-(1,1-dimethyl(ethyl)-oxazol-2-yl)vinyl]-2-yl pyrazole(X)

To a 0° C. solution of trifluoroacetic acid (2 ml) was added 1.26 g ofIX, the reaction was allowed to warm to room temperature, and stirred atroom temperature for 30 minutes. The solvent was removed and the yellowresidue was dried under high vacuum overnight to give the desiredproduct in 84% yield. The desired product was used in the next stepwithout further purification.

I. Preparation ofN,N-[5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylAmine (XI)

232 mg of the crude olefin X was taken up in 1 ml of dry pyridine andtaken to dryness under vacuum. The oil was co-evaporated with dryacetonitrile to yield a pale yellow solid. The solid was taken up in 15ml of chloroform and 1.3 ml of triethyl amine. To this solution wasadded 383 mg of 2,6-difluorophenyl isocyanate and the reaction heated toreflux overnight under a drying tube. The resulting product was purifiedby HPLC using a YMC S5 (ODS 20×100 mm) column to yield 89 mg of pureproduct. (M+H)⁺=388. HPLC RT=4.22 min (YMC S5 ODS column, 4.6×50 mm;10-90% MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nM detection).

EXAMPLE 2N-[5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(Methyl)propanamide(XV)

A. Preparation of5-Carboxymethyl-3-(2-methyl(ethyl)carbonylamino)pyrazole (XII)

A solution consisting of 398 mg of 5-carboxymethyl-3-aminopyrazole(synthesized as described above for the 5-ethyl analog, by use of MeOHrather than EtOH in the esterification) and 440 mL of pyridine in 5 mlof anhydrous dioxane was cooled to 0° C. To this solution was slowlyadded 621 mL of 2-methylpropanoyl chloride. The reaction was allowed tostir at room temperature for two hours, diluted with 25 ml of chloroformand extracted once against 15 ml of 1 M HCl followed by 15 ml ofsaturated NaHCO₃. The organic layer was isolated and dried over MgSO₄and the solvent was removed under vacuum. The product was used in thenext step without further purification.

B. Preparation of5-Hydroxymethyl-3-(2-methyl(ethyl)carbonylamino)pyrazole (XIII)

A solution of 787 mg of crude XII in 10 ml of dry THF was cooled to 0°C. in an ice bath. To this mixture was added 14 ml of 1 M LiAlH₄ in THFwhile maintaining the temperature at 0° C. The reaction was allowed tostir at 0° C. for 6.5 hours at which point the solution was poured over100 g of crushed ice. Once the ice had melted, the solution was broughtto pH 7 by addition of HCl. The water layer was filtered to removealuminum salts and the filtrate was extracted three times with 75 ml ofethyl acetate. The organic layer was collected, dried over MgSO₄ andevaporated to an orange solid. The solid was purified on silica gel togive 79 mg of the alcohol (15%).

C. Preparation of5-Carboxaldehyde-3-(2-methyl(ethyl)carbonylamino)pyrazole (XIV)

A solution consisting of 79 mg of XIII and 150 mg of MnO₂ in 10 ml ofacetone was heated to reflux for 20 hours. The mixture was filteredthrough a cake of celite and the cake was washed with excess acetone.The solvent was removed to yield a brown solid, which was purified bysilica gel to yield 58 mg (75%) of the desired product in pure form.

D. Preparation ofN-(E)-[5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]-2-(methyl)propanamide(XV)

A solution of 110 mg of (5-t-butyl-oxazol-2-yl)methylphosphonic aciddiethyl ester in 1 ml of anhydrous THF under N₂ was cooled to 0° C. and800 ml of a 1 M THF solution of potassium tertbutoxide was added. Thereaction was allowed to stir at 0° C. for 30 minutes at which time theanion solution was syringed into a solution of 29 mg of XIV dissolved in1 ml of anhydrous THF under N₂. The reaction was allowed to stir at roomtemperature overnight. The solution was evaporated onto silica gel andthe product was isolated by silica gel chromatography to give 16.6 mg(35%) of the desired product as a tan solid. (M+H)⁺=303. HPLC RT=3.51min (YMC S5 ODS column, 4.6×50 mm; 10-90% MeOH/H₂O gradient,+0.1% TFA; 4mL/min, 220 nM detection).

EXAMPLE 3N-(Z)-[5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide(XVI)

A solution of 32 mg of XV (described in Example 2) dissolved in 700 μlof acetone in a glass tube was irradiated for 3 days with an UV lamp toyield a 97% conversion to the E olefin isomer. The product was purifiedby silica gel chromatography to give 21 mg of the desired product.(M+H)⁺=303. HPLC RT=3.72 min (YMC S5 ODS column, 4.6×50 mm; 10-90%MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nM detection).

EXAMPLE 4N,N-[(E)-5-(2-(5-(1,1-Dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylAmine (XVII)

The above compound was prepared following procedures analogous to thosedescribed in Example 1. (M+H)⁺=388. HPLC RT=3.62 min (YMC S5 ODS column,4.6×50 mm; 10-90% MeOH/H₂O gradient,+0.1% TFA; 4 mL/min, 220 nMdetection).

We claim:
 1. A compound of the formula:

and pharmaceutically acceptable salts thereof wherein: R¹ is R², COR³,CONH₂, CONR²R³, COOR³, or SO₂R³; R² is H, alkyl, cycloalkyl,heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl, aryl,heteroaryl, arylalkyl, heteroarylalkyl, or heteroarylium; R³ is alkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl,aryl, heteroaryl, arylalkyl, heteroarylalkyl; A is

 wherein i, j=0 or 1 but cannot both be 1, and Y is optionallysubstituted ethylene, alkene, alkyne, or any 2 adjacent carbon atoms ofa cycloalkyl or cycloheteroalkyl ring of 3-7 atoms; R⁴ is R⁹; each R⁵,R⁶, R⁷, R⁸ is, independently, H, alkyl, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl,heteroarylalkyl, halo, or hydroxy, alkoxy, amino, NR¹⁵R¹⁶, thio, oralkylthio with the proviso that only one such heteroatom group is bondedto any one carbon atom; R⁹ is

 wherein Z is O or S; each R¹⁰, R¹¹ is, independently, H, alkyl,cycloalkyl, heterocycloalkyl, cycloalkylalkyl, heterocycloalkylalkyl,aryl, heteroaryl, arylalkyl, heteroarylalkyl, halo, hydroxy, alkoxy,alkylcarbonyloxy, carboxy, alkyloxycarbonyl, amino, NR¹³R¹⁴, carbamoyl,ureido, thio, or alkylthio; and each R¹², R¹³, R¹⁴, R¹⁵, and R¹⁶ is,independently, H, alkyl, cycloalkyl, heterocycloalkyl, cycloalkylalkyl,heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl.2. The compound of claim 1 wherein: R¹ is R², COR³, or CONR²R³; R² is H,alkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; R³ is alkyl,aryl, heteroaryl, arylalkyl or heteroarylalkyl; A is

 wherein i=j=0; Y is optionally substituted ethylene, alkene, or any twoadjacent carbon atoms of a cycloalkyl ring; R⁴ is R⁹; each R⁵, R⁶, R⁷,R⁸ is, independently, H, or alkyl; R⁹ is

 wherein Z is O; and each R¹⁰, R¹¹ is, independently, H, or alkyl. 3.The compound of claim 1 wherein: R¹ is CONR²R³; R² is H, alkyl,heteroaryl, arylalkyl, or heteroarylalkyl; R³ is aryl; A is

 wherein i, j=0 or 1 but cannot both be 1, and Y is an optionallysubstituted ethylene or alkene; R⁴ is R⁹; each R⁵, R⁶, R⁷, R⁸ is,independently, H, or alkyl; R⁹ is

 wherein Z is O; and each R¹⁰, R¹¹ is, independently, H, or alkyl. 4.The compound of claim 1 wherein: R¹ is R²; R² is alkyl, cycloalkyl,heterocycloalkyl, aryl, heteroaryl, arylalkyl, or heteroarylalkyl; A is

 wherein i, j=0 or 1 but cannot both be 1, and Y is an optionallysubstituted ethylene or alkene; each R⁵, R⁶, R⁷, R⁸ is, independently,H, or alkyl; R⁴ is R⁹; R⁹ is

 wherein Z is O; R¹⁰ is alkyl; and R¹¹ is H.
 5. The compound of claim 1wherein: R¹ is COR³; R³ is alkyl, cycloalkyl, heterocycloalkyl,cycloalkylalkyl, heterocycloalkylalkyl, aryl, heteroaryl, arylalkyl, orheteroarylalkyl;

 wherein i, j=0 or 1 but cannot both be 1, and Y is an optionallysubstituted ethylene, alkene or alkyne; R⁴ is R⁹; each R⁵, R⁶, R⁷, R⁸is, independently, H, or alkyl; R⁹ is

 wherein Z is O; R¹⁰ is alkyl; and R¹¹ is H.
 6. A compound selected fromthe group consisting of:N,N-[(Z)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylamine;N-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide;N-(Z)-[5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2-(methyl)propanamide;andN,N-[(E)-5-(2-(5-(1,1-dimethyl(ethyl))-oxazol-2-yl)vinyl)pyrazol-3-yl]2,6-difluorophenylaminocarbonylamine.
 7. A pharmaceutical composition comprising a compound of claim 1and a pharmaceutically acceptable carrier.
 8. A pharmaceuticalcomposition comprising a compound of claim 1 in combination with apharmaceutically acceptable carrier and an anti-cancer agent formulatedas a fixed dose.
 9. A pharmaceutical composition comprising a compoundof claim 1 in combination with a pharmaceutically acceptable carrier anda modulator of p53 transactivation formulated at a fixed dose.
 10. Amethod of inducing apoptosis which comprises administering to amammalian specie in need thereof an effective apoptosis inducing amountof a compound of claim
 1. 11. A method of inhibiting protein kinaseswhich comprises administering to a mammalian specie in need thereof aneffective protein kinase inhibiting amount of a compound of claim
 1. 12.A method of inhibiting cyclin dependent kinases which comprisesadministering to a mammalian specie in need thereof an effective cyclindependent kinase inhibiting amount of a compound of claim
 1. 13. Amethod of inhibiting cdc2 (cdk1) which comprises administering to amammalian specie in need thereof an effective cdc2 inhibiting amount ofa compound of claim
 1. 14. A method of inhibiting cdk2 which comprisesadministering to a mammalian specie in need thereof an effective cdk2inhibiting amount of a compound of claim
 1. 15. A method of inhibitingcdk3 which comprises administering to a mammalian specie in need thereofan effective cdk3 inhibiting amount of a compound of claim
 1. 16. Amethod of inhibiting cdk4 which comprises administering to a mammalianspecie in need thereof an effective cdk4 inhibiting amount of a compoundof claim
 1. 17. A method of inhibiting cdk5 which comprisesadministering to a mammalian specie in need thereof an effective cdk5inhibiting amount of a compound of claim
 1. 18. A method of inhibitingcdk6 which comprises administering to a mammalian specie in need thereofan effective cdk6 inhibiting amount of a compound of claim
 1. 19. Amethod of inhibiting cdk7 which comprises administering to a mammalianspecie in need thereof an effective cdk7 inhibiting amount of a compoundof claim
 1. 20. A method of inhibiting cdk8 which comprisesadministering to a mammalian specie in need thereof an effective cdk8inhibiting amount of a compound of claim
 1. 21. A method for treatingproliferative diseases comprising administering to a mammalian specie inneed thereof a therapeutically effective amount of a composition ofclaim
 7. 22. A method for treating cancer comprising administering to amammalian specie in need thereof a therapeutically effective amount of acomposition of claim
 7. 23. A method for treating inflammation,inflamatory bowel disease, or transplantation rejection, comprisingadministering to a mammalian specie in need thereof a therapeuticallyeffective amount of a composition of claim
 7. 24. A method for treatingarthritis comprising administering to a mammalian specie in need thereofa therapeutically effective amount of a composition of claim
 7. 25. Amethod for treating proliferative diseases comprising administering to amammalian specie in need thereof a therapeutically effective amount of acomposition of claim
 8. 26. A method for treating cancer comprisingadministering to a mammalian specie in need thereof a therapeuticallyeffective amount of a composition of claim
 8. 27. A method for treatingproliferative diseases comprising administering to a mammalian specie inneed thereof a therapeutically effective amount of a composition ofclaim
 9. 28. A method for treating cancer comprising administering to amammalian specie in need thereof a therapeutically effective amount of acomposition of claim
 9. 29. A method for the treatment of a cyclindependent kinase-associated disorder, comprising administering to asubject in need thereof an amount effective therefor of at least onecompound of claim
 1. 30. A method for treating chemotherapy-inducedalopecia, chemotherapy-induced thrombocytopenia, chemotherapy-inducedleukopenia or mucocitis which comprises administering to a mammalianspecie in need thereof a therapeutically effective amount of a compoundof claim 1.